The present invention generally relates to cell cycle checkpoint kinases which are essential to cellular DNA damage responses and coordinating cell cycle arrest. The checkpoint kinases play a role in the surveillance and response to DNA damage. The damage may result from external or internal forces. Such forces include but are not limited to errors in replication, DNA base damage, DNA strand breaks, or exposure to radiation or cytotoxic chemicals. These checkpoint kinases are integral in the regulatory pathways leading to cell cycle arrest and apoptosis following DNA damage, giving the cell notice and time to correct lesions prior to the initiation of replication and chromosome separation. The present invention more specifically relates to the isolation and purification of the catalytic domain of the human effector checkpoint protein kinase (hChk1) and its use in the discovery, identification and characterization of inhibitors of same.
Cell growth, division and death is essential to the life cycle of multi-celled organisms. These processes and their regulation are strikingly similar across all eukaryotic species. Somatic cell division consists of two sequential processes: DNA replication followed by chromosomal separation. The cell spends most of its time preparing for these events in a growth cycle (interphase) which in turn consists of three subphases: initial gap (G1), synthesis (S), and secondary gap (G2). In G1, the cell, whose biosynthetic pathways were slowed during mitosis, resumes a high rate of biosynthesis. The S phase begins when DNA synthesis starts and ends when the DNA content of the nucleus has doubled. The cell then enters G2, which lasts until the cell enters the final phase of division, mitotic (M). The M phase begins with nuclear envelope breakdown, chromosome condensation and formation of two identical sets of chromosomes which are separated into two new nuclei. This is followed by cell division (cytokinesis) in which each nuclei is separated into two daughter cells, which terminates the M phase and marks the beginning of interphase for the new cells.
The sequence in which the cell cycle events proceed is tightly regulated such that the initiation of one cell cycle event is dependent upon the successful completion of the prior cell cycle event. The process of monitoring genome integrity and preventing cell cycle progress in the event of DNA damage has been described as a xe2x80x98cell cycle checkpointxe2x80x99 (Hartwell, L H et al., Science, 246:629-634 (1989); Weinert et al., Genes and Dev., 8:652 (1994)]. Cell cycle checkpoints consist of signal transduction cascades which couple DNA damage detection to cell cycle progression. Checkpoints are control systems that coordinate cell cycle progression by influencing the formation, activation and subsequent inactivation of the cyclin-dependent kinases. Checkpoint enzymes are responsible for maintaining the order and fidelity of events of the cell cycle by blocking mitosis in response to unreplicated or damaged DNA. These enzymes prevent cell cycle progression at inappropriate times, maintain the metabolic balance of cells while the cell is arrested and in some instances can induce apoptosis (programmed cell death) when the requirements of the checkpoint have not been met (O""Connor, P M, Cancer Surveys, 29, 151-182 (1997); Nurse, P, Cell, 91, 865-867 (1997); Hartwell, L H et al, Science, 266, 1821-1828 (1994); Hartwell, L H et al., Science, 246, (1989), supra).
One series of checkpoints monitors the integrity of the genome. Upon sensing DNA damage, these xe2x80x9cDNA damage checkpointsxe2x80x9d block cell cycle progression in G1 and G2 phases, and slow progression through S phase (O""Connor, P M, Cancer Surveys, 29 (1997), supra; Hartwell, L H et al, Science, 266, (1994), supra). This action enables DNA repair to be completed before replication of the genome and subsequent separation of this genetic material into new daughter cell takes place.
Various mutations associated with malignancy affect the cancer cells ability to regulate checkpoints, allowing cells with DNA damage the increased likelihood to continue replicating and to escape damage-mediated apoptosis These factors contribute to the genomic instability which drives the genetic evolution of human cancers and contributes to the resistance of cancer cells to most current chemotherapy and radiotherapy intervention.
Due to abnormalities in the p53 tumor suppressor pathway, most cancer cells lack a functional G1 checkpoint control system. This makes them particularly vulnerable to abrogation of the last remaining barrier protecting them from the cancer killing effects of DNA damaging agents: the G2 checkpoint. The G2 DNA damage checkpoint ensures maintenance of cell viability by delaying progression into mitosis in cells that have suffered genomic damage. The G2 checkpoint is controlled by cell cycle checkpoint pathways which inhibit mitosis if previous events are incomplete or if the DNA is damaged. This regulation control system has been conserved from yeast to humans. Important in this conserved system is a kinase, Chk1 (or p56Chk1), which transduces signals from the DNA damage sensory complex to inhibit activation of the cyclin B/Cdc2 kinase which promotes mitotic entry (Peng, C Y et al, Science, 277, 1501-1505 (1997); Sanchez Y, et al., Science, 277, 1497-1501 (1997); Walworth, N et al., Nature, 363(6427), 368-71 (May 27, 1993); al-Khodairy et al., Mol Biol Cell, 5(2):147-60 (Febuary 1994); Carr et al., Curr Biol., 5(10): 1179-90 (Oct. 1, 1995)). The repair checkpoint kinase, Chk1, regulates Cdc25, a phosphatase that activates Cdc2. Thus, Chk1 serves as the direct link between the G2 checkpoint and the negative regulation of Cdc2.
Inactivation of Chk1 has been shown to both abrogate G2 arrest induced by DNA damage inflicted by either anticancer agents or endogenous DNA damage, as well as, result in preferential killing of the resulting checkpoint defective cells (Nurse, P, Cell, 91, (1997), supra; Weinert, T, Science, 277, 1450-1451 (1997); Walworth, N et al., Nature, 363, (1993) supra; al-Khodairy et al., Molec. Biol. Cell, 5, (1994), supra; Wan, S et al., Yeast, 15(10A), 821-8 (July 1999)).
The fact that cancer cells have also been shown to be more vulnerable to G2 checkpoint abrogation has encouraged the pursuit of G2 checkpoint abrogating drugs (Wang, Q et al., PNAS 96: 3706-3711 (1999); Fan, S et al., Cancer Res., 55, 1649-1654 (1995); Powell, S N et al., Cancer Res., 55, 1643-1648 (1995); Russell, K J et al., Cancer Res., 55, 1639-1642 (1995); Wang, Q et al., J Natl Cancer Inst., 88, 956-967 (1996)). Such checkpoint abrogating drugs could improve the killing of tumors exposed to DNA damaging events including that inflicted by therapeutic agents, hypoxic-stress induced because of a limited blood supply (anti-angiogenic agents), or endogenous DNA damage arising as a consequence of a cancer cell""s inherent genomic instability. Selective manipulation of checkpoint control in cancer cells can afford broad utilization in cancer chemotherapeutic and radiotherapy regimens and may in addition, offer a common hallmark of human cancer xe2x80x9cgenomic instabilityxe2x80x9d to be exploited as the selective basis for the destruction cancer cells.
A number of lines of evidence place Chk1 as a pivotal target in DNA damage checkpoint control. However, Chk1 is a difficult enzyme to study because the full length protein is not the most active form of Chk1. While others have examined the nucleotide and amino acid sequence of the full-length checkpoint kinase and estimated the location of the kinase domain, there is a need for the isolation and purification of the kinase domain of Chk1and the maintenance of its catalytically active conformation.
The generation, kinetic characterization, and structure determination of the kinase domain of the human Chk1 protein is disclosed herein. The domain begins between residues 1 and 16 and terminates between residues 265 and 291 of the full length protein [SEQ ID NO. 2] which comprises 476 amino acids. The domain preferably extends from residues 1-265, more preferably from residues 1-289.
The invention relates to an isolated, purified polynucleotide which encodes the active conformation of the human Chk1 kinase or an active kinase analog thereof. The polynucleotide may be natural or recombinant.
The invention also relates to an isolated, soluble catalytically active polypeptide comprising the active conformation of the human Chk1 kinase or an active kinase analog thereof.
The invention encompasses both the polypeptide per se as well as salts thereof. As discussed in detail below, a high salt concentration (about 500 mM) in the buffer is used herein to prevent aggregation of peptide during purification and storage.
The invention also relates to a crystal structure of the human Chk1 kinase in the active conformation resolved to at least 2.5 xc3x85, preferably 2.0 xc3x85, more preferably 1.7 xc3x85. This structure provides a three-dimensional description of the target (human Chk1) for structure-based design of small molecule inhibitors thereof as therapeutic agents.
The invention further relates to an expression vector for producing catalytically active human Chk1 kinase in a host cell.
The invention further relates to a host cell stably transformed and transfected with a polynucleotide encoding of the human Chk1 kinase, or fragment thereof, or an active kinase analog thereof, in a manner allowing the expression of the human Chk1 kinase in the active configuration.
The present invention further discloses methods for screening candidate compounds using the molecular structure of the x-ray crystallography data to model the binding of candidate compounds.
The invention further provides a method for designing and screening potentially therapeutic compounds for the treatment of hyper-proliferative or diseases related to proliferation, including but not limited to cancer and HIV infection. The putative therapeutics can be screened for activities such as (1) potentiation of the cytotoxicity of DNA damaging agents such as synthetic or natural chemotherapeutic agents and ionizing or neutron radiation; (2) enhancement of the cytotoxicity of DNA synthesis inhibitors including antimetabolites, DNA chain terminators, or other mechanisms that would lead to the inhibition of DNA synthesis; (3) enhancement of the cytotoxicity of hypoxia as would occur within tumors due to a limited blood supply; and (4) inhibition of the ability of HIV to arrest cell cycle progression such as that induced by the VPR protein. Compounds that inhibit human Chk1 kinase activity or abrogate the G2 checkpoint can be used to treat or prevent the hyperproliferation associated with cancer and HIV.
The present invention provides methods for identifying potential inhibitors of the human Chk1 protein kinase by de novo design of novel drug candidate molecules that bind to and inhibit human Chk1 protein kinase activity, or that improve their potency. The x-ray crystallographic coordinates disclosed herein, allow generation of 3-dimensional models of the catalytic site and the drug binding site of the human Chk1 protein. De novo design comprises of the generation of molecules via the use of computer programs which build and link fragments or atoms into a site based upon steric and electrostatic complementarily, without reference to substrate analog structures. The drug design process begins after the structure of the target (human Chk1 kinase) is solved to at least a resolution of 2.5 xc3x85. Refinement of the structure to a resolution of 2.0 xc3x85 or better with fixed water molecules in place provides more optimal conditions to undertake drug design.
The invention further provides a method for computational modeling of the kinase domain of human Chk1, such a model being useful in the design of compounds that interact with this domain. The method involves crystallizing the Chk1 kinase in the catalytically active configuration; resolving the x-ray structure of said active kinase, particularly the kinase domain and binding site of active Chk1; and applying the data generated from resolving the x-ray structure to a computer algorithm capable of generating a three dimensional model of the kinase domain and binding site suitable for use in designing molecules that will act as agonists or antagonists to the polypeptide. An iterative process can then be applied to various molecular structures using the computer-generated model to identify potential agonists or antagonists of the Chk1 kinase. Inhibitors of the kinase can serve as lead compounds for the design of potentially therapeutic compounds for the treatment of diseases or disorders associated with hyperproliferation or related to proliferation, such as cancer and HIV.
The invention further provides a process where the human Chk1 protein kinase is modified by deletion of the C-terminal portion of the protein so as to impart favorable physical characteristics of the resulting polypeptide. The kinase domain is suitable for analysis by nuclear magnetic resonance, high throughput screening, biochemical characterizations, x-ray crystallography, colorimetry and other diagnostic means. The most preferred deletion fragment extends from residue 1 to residue 289.
The invention further provides screening methods for use in the drug design process of potential agents to the human Chk1 protein kinase by de novo design of novel drug candidate molecules with potentially nanomolar potencies. The x-ray crystallographic coordinates disclosed based on the kinase domain of the human Chk1 protein will allow the generation of 3-dimensional models of the active binding sites of the human Chk1 protein.
The invention further provides a method for rapidly screening compounds to identify those compounds that inhibit Chk1 kinase or core structure for further Chk1 inhibitor design. The high throughput-screening assay is capable of being fully automated on robotic workstations. The assay may be radioactive. However, in a preferred embodiment the assay is a non-radioactive ELISA. In a more preferred embodiment, the assay is an ELISA that utilizes a novel antibody, rabbit anti-phosphosyntide, to specifically detect the product of the Chk1 kinase reaction in which biotin-syntide is the substrate. However, the basis of the assay includes the ability to use other substrates detectable by anti-phospho-peptide/protein antibodies. The assay may be used to screen large collections of compound libraries to discover Chk1 kinase inhibitors and potential lead compounds for the development of Chk1 kinase selective anticancer compounds. The assay finds utility in the screening of other syntide substrate kinase reactions involving kinases of analogous activity to Chk1.